Four previously undescribed naphthohydroquinone dimers named rubipodanones A-D, together with 19 known quinones containing three known napthohydroquinone dimers named rubioncolin C, methyl 5-hydroxy-dinaphtho[1,2-2′,3′]furan-7,12-dione-6-carboxylate and rubialatin B, were isolated from the roots and rhizomes of Rubia podantha. Their structures and absolute configurations were determined mainly by NMR, X-ray diffraction, and computational methods. Rubipodanones C and D, the glycosides of rubipodanone A and a pair of C-3 epimer, are the first identified dimeric napthohydroquinone glycosides from the Rubia plants. All naphthohydroquinone dimers were evaluated for their cytotoxicities against ten tumor cell lines and effects on the tumor-associated NF-κB signaling pathway, and rubioncolin C showed the best cytotoxicity with IC50 value of 1.53 μM and NF-κB inhibitory activity with IC50 value of 2.97 μM. These results also demonstrated that the key roles of C-3 configuration and sugar group for biological activities of rubipodanone C.